Validating neural spheroids as HTS platform for the discovery and validation of new treatments for OUD. Acquired neural 3D spheroid cultures from Stemonix in 96-well plates as a high throughput system to test compounds. Development assay outputs including calcium flux (FLIPR), neurotransmitters (LC-MS, MALDI in development), and high content imaging (eg, neuronal populations, synapse plasticity, in development). The goal is to develop OUD assay signatures to assess effect of compound/targets on OUD models a acute (min-hour) and chronic (weeks) (eg opioid conditioning, a period of drug abstinence, and a subsequent return to drug administration). These self-organized brain organoids are generated from NPC and include mature cortical neurons (Cortical neurons include both excitatory (glutamatergic) and inhibitory (GABAergic)) and astrocytes making them relevant to study opioid-like activity signatures using detection methods commonly used in HTS, including fluorescence imaging, calcium flux and multielectrode arrays (MEA), all readers already available at NCATS. This year, we demonstrated that these neural spheroids have synchronized calcium waves, and using this readout, we have implemented a focused screen of 80 compounds modulating targets related to addiction, and shown that the assay platform is robust for HTS. We are also exploring chronic addiction of opioids perturb calcium waves in these spheroids to be able to model opioid withdrawal and relapse effects. Developing ex vivo brain assembloids that includes brain microvasculature with brain endothelial cells in a microenvironment made up of astrocytes, pericytes and neuronal cells. This year, we have shown that we can 3D bioprint a neurovascular unit with brain ECs, pericytes and astrocytes. We are in the process of including neuronal cells and demonstrate physiological activity. We are also working on developing a new multiwell plate platform that will enable perfusion though the vasculature. Developing a HT physiologically and pharmacologically relevant blood brain barrier (BBB). A physiological relevant Blood Brain Barrier (BBB) Model is being developed to study drug penetration in the brain as well as to study the effects of drugs on the physiology of the BBB in the context of pain and addiction. We are working towards the production of a 96-well transwell BBB model, comparing human primary and human iPSC-derived sources of human brain endothelial cells (ECs) and using transepithelial electrical resistance (TEER) measurements as a marker indicating tight junction and barrier formation. This year we have demonstrated that we can generate iPSC-derived brain ECs and produce a BBB in 96-well transwell with physiological relevant TEER values. We are also exploring microfluidics platforms such as the Mimetas platforms to develop a HT BBB model. Constructing ex vivo neuronal circuits to model the VTA addiction/rewards system. A simplified In vivo reward circuit, for example the ventral tegmental area, is being constructed by bioprinting for testing the effects of compounds in reward and addiction neuronal circuits. We are exploring the use of optogenetics (opsin proteins) in iPSC-derived neurons as al method to control neuronal stimulation. We are using of MEA and fluorescence biosensors as assay detection technologies Developing an innervated skin tissue model as an assay platform for peripheral pain. We are collaborating with the NCATS SCTL to use their iPSC-derived sensory neurons and our developed skin biofabrication protocols to establish a innervated skin model to be used as an assay platform for peripheral pain.